Genetic structure of the population of wild-growing vines of the Utrish Nature Reserve

Grapes are one of the most common agricultural crops in the world. Currently, the analysis of genotypes directly at the DNA level is considered to be the most accurate method for studying the plant gene pool. The study of wild vines and ancient varieties in various regions of viticulture is an important direction of research in this field. The purpose of this work was to study the population of wild grapes growing on the territory of the Utrish Nature Reserve on the Black Sea coast of Krasnodar Region. The territory of the reserve is of interest as it is a site of ancient settlements, and the environmental conditions are suitable for the growth of wild grapes. During the survey of the territory, 24 samples of wild grapes were found, which were described according to the main morphological characteristics and analyzed by the molecular genetic method. The found vines were genotyped using 15 DNA markers, including nine commonly used for DNA fingerprinting (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79) and VVIb23, which allows determining hermaphrodite and dioecious vines. Statistical processing of microsatellite loci polymorphism data was carried out using the GenAlEx 6.5 program. The genetic relationships of the studied vines were evaluated using the PAST 2.17c program. The samples were found to be morphologically and genetically polymorphic. The number of alleles identified in the sample varied from 5 to 18 and averaged 8 alleles per locus. Statistical processing of DNA analysis data made it possible to identify two genetically different populations among the wild discovered vines. An assessment of genetic similarity of the found vines with some local varieties of geographically close viticulture regions, rootstocks and representatives of Vitis sylvestris from other territories was made. One of the populations found in the Utrish Nature Reserve is close to a number of V. sylvestris genotypes, the DNA profiles of which are presented in the Vitis International Variety Catalogue.


Introduction
Grapes are one of the most widespread agricultural crops in the world. The most significant, both economically and sociohistorically, is the species Vitis vinifera L. This species of the genus Vitis L. (family Vitaceae), originating from Eurasia, sup posedly appeared about 65 million years ago (This et al., 2006). Currently, two subspecies are distinguished within the species, V. vinifera L. subsp. sylvestris (Gmel.) Hegi, which includes wild populations, and V. vinifera subsp. sativa (DC.) Hegi (or subsp. vinifera), which includes cultivars.
Wild and cultivated vines differ in a number of features, including their reproductive biology: wild grapevines are dio ecious and cross-pollinate, while the cultivated vines are mostly hermaphrodite and self-pollinating. The domestication of grapes, which occurred about 8 thousand years ago, is closely associated with the emergence of winemaking, although it is still not known for certain which process preceded the other. The Middle East and the Caucasus are considered to be the initial centers of domestication of V. vinifera. The earliest evidence of wine production from 7,400-7,000 BC is found in Iran (McGovern, 2004). Seeds of domesticated grapes, about 8,000 years old, have also been found in Georgia and Turkey. However, Neolithic seed remains found in Western Europe also suggest grape exploitation during this time, and wild form seed remains have been found at Bronze Age sites in France (This et al., 2006).
Wild grape populations are currently represented by wild vines of V. vinifera cultivars and a wild subspecies. The study of wild grapevines in the ancient regions of grape cultivation has been actively conducted in recent years at the molecular genetic level (Doulati-Baneh et al., 2015;Gorislavec et al., 2017;De Michele et al., 2019;Margaryan et al., 2019;Cunha et al., 2020;Zdunić et al., 2020;Kupe et al., 2021;Lukšić et al., 2022).
The study of the local gene pools of various viticulture regions (including native varieties and wild specimens) at the DNA level makes it possible to more fully assess the genetic diversity of varieties and vines, to identify closer and more distant genotypes. Wild exemplars of agricultural crops are also significant for breeding as unique sources of genetic variability (Ellstrand et al., 2010).
The territory of the Utrish Nature Reserve on the Black Sea coast of the Krasnodar Territory is of interest for this kind of research, since it is a site of ancient settlements, and the ecological conditions of the territory are suitable for the growth of wild vines (Chernodubov, Rudenok, 2015).

Materials and methods
Expeditionary research to search for wild grapevines, study the ecological conditions of their habitats and morpho-biological features was carried out for three years (2019-2021) on the ter ritory of the Utrish Nature Reserve (Krasnodar Territory). The reserve is located in the northwestern part of the Black Sea coast of the Western Caucasus, on the Abrau Peninsula. The climate is sub-Mediterranean, moderately warm.
For molecular genetic analysis, 24 samples of wild-growing grapes were selected. DNA samples were isolated from shoot apical parts of vine plants by a method based on the use of CTAB (cytyltrimethylammonium bromide) (Rogers, Bendich, 1985).
The study was carried out using the instrument park of the Center for Collective Use of Technological Equipment in the direction of "Genomic and postgenomic technologies" of the North Caucasian Federal Scientific Center for Horticulture, Viticulture, Wine-making.

Results and discussion
In the process of surveying the territory of the Utrish Nature Reserve, 24 samples of wild-growing grapes were found, the description of which according to the main morphological features is given in Table 1.
Polymorphism of morphological traits was noted both between populations from different sampling sites, and between plants within nominal populations, designated by us according to their places of growth on the territory of the reserve (plants were found in the locations of Atmacheva Shchel (A1-A3), Shirokaya Shchel (Sh1-Sh5), Lobanova Shchel (L1-L6), Vo dopadnaya Shchel (V1-V10) . The ecological and geographical characteristics of the places where grape plants were found are presented in Table 2.
DNA analysis of the found grape samples revealed different levels of polymorphism in the studied 15 loci -the number of identified alleles in the sample varied from 5 (VVMD25, VVMD27 and GF09-46) to 18 (UDV305) and amounted to an average of 8 alleles per locus (Table 3). The mean observed heterozygosity (Ho = 0.664) was lower than expected (He = 0.712). DNA marker analysis using VVIb23 showed that all wild vines were dioecious. Among the nine microsatellite loci, the data on polymorphism of which are used for DNA fingerprinting of grape genotypes, the most polymorphic was VVS2 (10 alleles were determined), the least -VVMD25, VVMD27 (5 types of alleles each) (see Table 3).
A similar situation was noted in the study of the diversity of wild vines in Armenia -the studied sample (77 samples) was also the most polymorphic in the locus of VVS2 -13 types of alleles, and the least in VVMD25 and VVMD27 (5 and 8 alleles were identified, respectively) (Margaryan et al., 2019). At the same time, in the study of Gorislavec S.M. and co-authors (2017), in which Crimean wild-growing vines were studied, the smallest polymorphism (5 types of alleles) was revealed at the VVS2 locus (Gorislavec et al., 2017).
When distributing genotypes in the space of the main coordinates, a group of samples from territory of the Vodopadnaya Shchel can be distinguished (Fig. 1). In general, all samples from this place are localized separately from others in the space of the main coordinates, while samples V1, V2, V3, V4, V9, V10 form a separate subgroup. Samples V6 and

V10
Genetic structure of the population of wild-growing vines of the Utrish Nature Reserve  V7 have very similar genotypes. Among other found samples, the complete coincidence of genotypes at the studied loci was determined in wild vines Sh3 and Sh4. Analysis of samples using DNA markers linked to the resistance genes to downy mildew Rpv3, Rp10 and powdery mildew Ren3, Ren9 did not reveal resistance loci in the genotypes of wild vines. The inclusion of these DNA markers in the study was carried out in order to study the polymorphism of wild grapes at the analyzed loci, as well as a tool for the possible identification of wild-growing vines of hybrid origin. The resistance determined by Ren3, Ren9 and Rpv3 is in herited from North American grape species (V. riparia, V. rupestris, V. labruska, V. lincecumii), Rpv10 -from V. amurensis.
DNA fingerprints of the studied wild-growing vines by 9 SSR loci standard for the identification of grape genotypes were checked for a coincidence in the catalog of grape varieties' DNA profiles of the international database VIVC (VIVC, 2022). No coincidences were found. To analyze the genetic similarity, DNA profiles of varieties belonging to geogra-phically close regions of viticulture, where there are local varie ties, some of which may have originated from wild grapevines growing earlier in these territories, were included into the research. For comparison, the identified DNA profiles of V. syl vestris genotypes from different geographical locations (Israel, Tunisia, France, Armenia, Turkey) presented in VIVC were also used. The study also included a group of rootstock varieties of complex interspecific origin with the largest share of the genetic contribution of North American grape species in order to exclude the presence of rootstock varieties among the found wild vines, which are characterized by high adaptability to various abiotic and biotic stress factors. The number of clusters equal to 7 (K=7) was used for Bayesian analysis. The results of the analysis are shown in Figure 2.

Conclusion
Based on the above, we can conclude that the samples of wild grapes of the Utrish Nature Reserve selected during the expedition are represented by two hypothetical populations (expressed as clusters 4 and 5). There are transitional forms between the two populations. And if the first nominal population (cluster 4) is localized on the Vodopadnaya Shchel territory, then representatives of the second population (cluster 5) are found at all expeditionary points of sampling of plant material. A genetic relationship was also established between the second nominal population and some genotypes of V. sylvestris and the Georgian variety Chkhaveri (45), with varying degrees of probability included in cluster 5. Thus, it can be assumed that at least part of the genotypes found in the Utrish Nature Reserve are close to the genotypes of the subspecies V. sylvestris presented in the VIVC international database. Samples from the hypothetical first population (primarily localized -Vodopadnaya Shchel) are genetically different from other vines and are predominantly allocated to cluster 4. An insignificant contribution of cluster 4 was noted in the genotypes from the gene pool of Georgian and Crimean local varieties (44 -Tsolikouri, and 48 -Kefesia), and also in two accessions of V. sylvestris (61 -Khedhayria (Tunisia), 65 -Sylvestris Guemuelduer 104-64 (Turkey)). The genetic contribution of American rootstock varieties to the wild grape population of the Utrish Nature Reserve has not been identified.